Feature Articles

Bringing GPCR Research to the Fore

The group of seven transmembrane-spanning G protein-coupled receptors (GPCRs) represents the largest superfamily of receptors in the human genome. GPCRs are involved in virtually all physiological processes and represent the targets for at least 30% of all current medicines. GPCRs sense molecules outside the cell and, in response, activate intracellular signal transduction pathways and, ultimately, cellular responses.

Stephen Rees, Ph.D., vp of screening sciences and management at AstraZeneca, described the results of a recent effort to identify native ligands for orphan GPCRs. Such efforts are quite important, Dr. Rees asserted, because “GPCRs are the most important target class for the discovery of molecules for pharmacological intervention in disease, both in terms of the proportion of marketed drugs with activity against this target class and in terms of industry revenue.” It therefore seems likely that many of these orphan GPCRs will also prove to be of medical significance.

Despite extensive efforts, Dr. Rees said, there remain over 100 GPCRs for which the native ligand has not been conclusively identified. Ligand identification would help enable target validation and would also enable drug screening assays for the GPCRs.

While Dr. Rees was at GlaxoSmithKline he was involved in a collaboration with DiscoveRx and Medical Research Council Technology (MRCT) to screen 90 orphan GPCRs against a comprehensive panel of 10,000 known bioactive molecules for their ability to interact with the GPCRs to selectively regulate the beta-arrestin signaling cascade. This method was chosen because it is relatively simple to measure, and it was believed that many, if not all, GPCRs affect this signaling cascade.

The result of the study was disappointing, Dr. Rees reported, as native ligands were identified for only 3 of the 90 orphan GPCRs. Dr. Rees said that this may indicate that there may be only a small number of GPCRs that selectively regulate the beta-arrestin signaling cascade, and that ligands for the remaining orphan receptors remain to be discovered. Nevertheless, native ligands for three previously orphan GPCRs were identified, and two of these GPCRs are now the subjects of ongoing drug discovery efforts at MRCT.

Oncogenic and Pro-Metastatic Signaling Circuitries

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Researchers at the University of Wurzburg have studied cell receptors using fluorophores in order to directly observe the receptors, how and where they move, how fast they turn on and off, and how they trigger cell reactions.

J. Silvio Gutkind, Ph.D., chief, oral and pharyngeal branch, National Institute of Dental and Craniofacial Research, NIH, emphasized the growing awareness that GPCRs can play key roles in cancer and that these molecules represent a new source of anticancer targets that may be exploited. In particular, he noted that in the last three years cancer genome sequencing results have revealed an unexpectedly high rate of GPCR gene mutations (5% to 30%) in some of the most prevalent human cancers.

In fact, a Genentech study published in 2009 indicated that approximately 20% of cancer mutations actually occur in G protein or GPCR genes. Activating mutations in Gαs genes have been observed in multiple adenomas and genes for both Gαq family members (GNAQ and GNA11) and also were recently identified in approximately 80% of uveal melanomas, where these genes are considered the driving melanoma oncogenes.

A direct link between GPCRs and viral-associated malignancies was previously established (1998–2003) by Dr. Gutkind and his colleagues with the identification of a constitutively active oncogenic GPCR (vGPCR) coded by the Kaposi’s sarcoma (KS)-associated herpes virus (KSHV) that is the infectious cause of KS.

Dr. Gutkind said that angioproliferative tumors induced by KSHV have been successfully treated with rapamycin, providing direct evidence of the clinical activity of mTOR inhibition in human cancers. However, prolonged mTOR inhibition in immunocompromised patients, such as those with AIDS-KS, may raise concerns.

Therefore, Dr. Gutkind’s group recently investigated whether KSHV oncogenes (specifically vGPCR) deploy cell type-specific signaling pathways activating mTOR and if such specific pathways could be targeted to halt KS development while minimizing immune-suppressive effects. The group determined that the PI3Kγ isoform is strictly required for signaling from the vGPCR oncogene to the Akt/mTOR signaling pathway. The group then generated genetic and pharmacological evidence that PI3Kγ may represent a suitable therapeutic target in KS.

Martin Lohse, M.D., professor of pharmacology at the University of Würzburg in Germany and chairman of the Rudolf Virchow Center, discussed his group’s efforts to carry out studies of single GPCR molecules. Dr. Lohse said that for a decade his group has studied cell receptors using fluorophores in order to directly observe the receptors, how and where they move, how fast they turn on and off, and how they trigger cell reactions. This work is now going in two directions—first in the direction of drug development and drug screening using optical tests, and secondly, in the direction of single-molecule studies.

Dr. Lohse believes the single-molecule studies have the potential to yield great insight into the workings of GPCRs. He drew the analogy with the progress allowed by the development of the patch-clamp technique in electrophysiology, which enabled the study of single-ion channels in cells and proved their involvement in fundamental cell processes.

Previously, scientists have only been able to look at the behavior of GPCRs in aggregate, and this behavior may be the sum of many different individual reactions.

Increasingly, Dr. Lohse said, the data on the overall behavior of GPCRs and signaling proteins are being complemented by studies of individual GPCRs and signaling proteins, which show distinct and specific behavior of individual proteins in terms of their mobility and protein-protein interactions.

These studies are made possible by using highly fluorescent labels combined with single particle tracking. Together, these experiments show that GPCRs have very distinct properties, ranging from receptors that act in strict linear chains to those that exist and function in large protein aggregates that allow complex interactions and signaling behavior.

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